Folding of a bacterial outer membrane protein during passage through the periplasm

Eppens, Elaine F

doi:10.1093/emboj/16.14.4295

Cited by 61 publications

(50 citation statements)

References 44 publications

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“…Complementation of the ␤-barrel by full-length mutant FhuA also favors the view that a fragment containing the cork is released in the periplasm and is incorporated into the ␤-barrel of FhuA⌬5-160. Much evidence exists that folding of outer membrane proteins starts in the periplasm and is completed in the outer membrane (14). Such a model is consistent with the data on FhuA, which may serve as a model system to work out the details of outer membrane protein biogenesis, since the cork and the ␤-barrel form separate domains and allow the stepwise assembly process to be unraveled.…”

Section: Discussionsupporting

confidence: 67%

“…According to current thinking, assembly of outer membrane proteins occurs in several steps. Two models have been discussed: (i) proteins are directly inserted into the outer membrane through contact sites between the cytoplasmic membrane and the outer membrane (1), and (ii) proteins pass through the periplasm on their way into the outer membrane (14,16,41). The data of this study favor the latter model, since separately synthesized cork is incorporated into separately synthesized ␤-barrel, which is most easily envisaged when the unfolded cork diffuses in the periplasm and finds its final conformation while being incorporated into the preformed ␤-barrel in the periplasm or during or after incorporation of the ␤-barrel in the outer membrane.…”

Section: Discussionmentioning

confidence: 99%

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In Vivo Reconstitution of the FhuA Transport Protein ofEscherichia coliK-12

et al. 2003

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The FhuA protein in the outer membrane of Escherichia coli actively transports ferrichrome and the antibiotics albomycin and rifamycin CGP 4832 and serves as a receptor for the phages T1, T5, and 80 and for colicin M and microcin J25. The crystal structure reveals a ␤-barrel with a globular domain, the cork, which closes the channel formed by the barrel. Genetic deletion of the cork resulted in a ␤-barrel that displays no FhuA activity. A functional FhuA was obtained by cosynthesis of separately encoded cork and the ␤-barrel domain, each endowed with a signal sequence, which showed that complementation occurs after secretion of the fragments across the cytoplasmic membrane. Inactive complete mutant FhuA and an FhuA fragment containing 357 N-proximal amino acid residues complemented the separately synthesized wild-type ␤-barrel to form an active FhuA. Previous claims that the ␤-barrel is functional as transporter and receptor resulted from complementation by inactive complete FhuA and the 357-residue fragment. No complementation was observed between the wild-type cork and complete but inactive FhuA carrying cork mutations that excluded the exchange of cork domains. The data indicate that active FhuA is reconstituted extracytoplasmically by insertion of separately synthesized cork or cork from complete FhuA into the ␤-barrel, and they suggest that in wild-type FhuA the ␤-barrel is formed prior to the insertion of the cork.The FhuA protein of Escherichia coli K-12 transports ferrichrome, the structurally related antibiotic albomycin, and the unrelated antibiotic rifamycin CGP 4832 across the outer membrane and serves as a receptor for colicin M, microcin J25, and the phages T1, T5, and 80 (7). Although extensive mutational analyses have been performed (6, 7, 33) and the crystal structure has been determined (15, 30), the molecular mechanism of FhuA transport is unclear.The protein consists of a ␤-barrel (residues 161 to 714) composed of 22 antiparallel ␤-strands that form a channel that is closed by a globular domain (residues 1 to 160), designated the cork or plug, that inserts from the periplasmic side into the ␤-barrel. Binding of ferrichrome elicits small, 1-to 2-Å movements of the cork domain relative to the ␤-barrel and a large 17-Å transition of E19 without opening the channel. Release of ferrichrome from the binding site formed by 10 amino acid residues located in the ␤-barrel and the cork, and opening of the channel, is thought to be triggered by interaction with the TonB protein, which requires the proton motive force of the cytoplasmic membrane to exert its action on FhuA. All FhuArelated activities, except infection by phage T5, depend on TonB.We previously reported that deletion of the cork (residues 5 to 160) results in a protein (FhuA⌬5-160) that still functions as an active TonB-dependent transporter and receptor (3,4,25). These experiments were performed with two fhuA mutant strains; mutant 41/2 contains a number of amino acid replacements and D348 is deleted (26), whereas PCR analysis indicated th...

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Section: Discussionsupporting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

In Vivo Reconstitution of the FhuA Transport Protein ofEscherichia coliK-12

et al. 2003

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“…This observation remains to be investigated in further detail. The in vivo mechanism of porins folding and insertion is not well established, but the implication of several patterns like lipopolysaccharides, periplasmic chaperones, and catalysts has been proposed (33)(34)(35)(36)(37). Black lipid membrane experiments represent a highly simplified model system where folded porins spontaneously insert into symmetric and planar lipid bilayers.…”

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confidence: 99%

Probing the Orientation of Reconstituted Maltoporin Channels at the Single-protein Level

Danelon

Brando

Winterhalter

2003

Journal of Biological Chemistry

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Recently we have shown that maltoporin channels reconstituted into black lipid membranes have pronounced asymmetric properties in both ion conduction and sugar binding. This asymmetry revealed also that maltoporin insertion is directional. However, the orientation in the lipid bilayer remained an open question. To elucidate the orientation, we performed point mutations at each side of the channel and analyzed the ion current fluctuation caused by an asymmetric maltohexaose addition. In a second series we used a chemically modified maltohexaose sugar molecule with inhibited entry possibility from the periplasmic side. In contrast to the natural outer cell wall of bacteria, we found that the maltoporin inserts in artificial lipid bilayer in such a way that the long extracellular loops are exposed to the same side of the membrane than protein addition. Based on this orientation, the directional properties of sugar binding were correlated to physiological conditions. We found that nature has optimized maltoporin channels by lowering the activation barriers at each extremity of the pore to trap sugar molecules from the external medium and eject them most efficiently to the periplasmic side.

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“…It is not known where the interaction occurs, after ␤-barrel incorporation into the outer membrane or in the periplasm with a periplasmic, partially folded ␤-barrel intermediate. Such intermediates have been described for several outer membrane porins (13,15).…”

Section: Vol 187 2005mentioning

confidence: 90%

Activities of the Serratia marcescens Heme Receptor HasR and Isolated Plug and β-Barrel Domains: the β-Barrel Forms a Heme-Specific Channel

et al. 2005

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The Serratia marcescens hemophore-specific outer membrane receptor HasR is a member of the TonB-dependent family of autoregulated receptors. It can transport either heme itself or heme bound to the hemophore HasA. On the basis of sequence and functional similarities with other TonB-dependent outer membrane receptors whose three-dimensional structures have been determined, a HasR structure model was proposed. The mature HasR protein comprises a 99-residue amino-terminal extension necessary for hasR transcription, followed by a plug domain of 139 amino acids and a ␤-barrel domain inserted in the outer membrane, the lumen of which is closed by the plug. This model was used to generate hasR deletions encoding HasR proteins with the native signal peptides but lacking either the N-terminal regulatory extension or encoding the plug or the ␤-barrel alone. The protein lacking the N-terminal extension, HasR ⌬11-91, was incorporated in the outer membrane and was fully functional for active uptake of free and hemophore-bound heme. The HasR ␤-barrel, ⌬11-192, was also incorporated in the outer membrane and bound the hemophore but expressed no active heme transport properties. The HasR plug remained in the periplasm. Coexpression of the plug and the ␤-barrel allowed partial plug insertion in the outer membrane, demonstrating that these two HasR domains interact in vivo. The ␤-barrel and the plug also interact in vitro. Nevertheless, the two domains did not complement each other to reconstitute an active TonB-dependent receptor for free or hemophore-bound heme uptake. Production of the ␤-barrel alone selectively increased passive diffusion of heme but not of other exogenous compounds. A mutation at histidine 603, which is required for heme uptake through the wild-type receptor, abolished heme diffusion, showing that HasR ⌬11-192 forms a specific heme channel.Gram-negative bacteria are surrounded by an outer membrane outside the peptidoglycan layer which delimits the periplasmic space, confining many extracytoplasmic enzymes to that space. The outer membrane also serves as a selective permeation barrier: it restricts permeability to lipophilic compounds and contains channels for nutrients and ions required for growth. These channels are made up of outer membrane proteins. They can be schematically grouped into three classes: passive nonspecific diffusion pores, specific channels, and active transporters. Diffusion channels allow entry of hydrophilic solutes smaller than 600 Da. Specific channels also allow some flow of small hydrophilic compounds and in addition bind their cognate substrates and allow their diffusion at micromolar concentrations. The three-dimensional structures of several outer membrane porins and specific channels have been determined. They form trimeric ␤-barrel structures, each subunit having a constricted central lumen of 12 Å diameter (see reference 27 for review).Larger compounds like siderophores and heme are actively transported through specific gated receptors. Despite the large diversity of their cog...

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Folding of a bacterial outer membrane protein during passage through the periplasm

Cited by 61 publications

References 44 publications

In Vivo Reconstitution of the FhuA Transport Protein ofEscherichia coliK-12

In Vivo Reconstitution of the FhuA Transport Protein ofEscherichia coliK-12

Probing the Orientation of Reconstituted Maltoporin Channels at the Single-protein Level

Activities of the Serratia marcescens Heme Receptor HasR and Isolated Plug and β-Barrel Domains: the β-Barrel Forms a Heme-Specific Channel

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